Equine arteritis virus (EAV) is a highly contagious virus that is spread via the respiratory or reproductive tract and causes persistent infections in horses and donkeys that are either asymptomatic or alternatively, quite severe like in those animals experiencing hemorrhagic fever or even miscarriages. EAV is a member of the Arteriviridae virus family, which also includes the lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV).
The biological and biophysical properties of EAV have frequently been the subject of experimental investigation, together with efforts to characterize EAV's viral pathogenesis and cell virus interactions. An overview of the genomic organization and transcriptional strategy of Arteriviruses is shown in FIG. 1.
EAV is characterized by a small (typically 60-65 nm in diameter) enveloped particle and has a 49S RNA genome that is a single-stranded, non-segmented, capped and polyadenylated message-sense RNA (12687 nucleotides; see den Boon et al., 1991, Equine arteritis virus is not a togavirus but belongs to the corona-virus-like superfamily. J. Virol. 65, 2910-2920; GenBank accession number: X53459).
The EAV genome is particularly infectious and contains at least eight open reading frames (ORF) including ORFs 1a, 1b, 2, 3, 4, 5, 6, and 7. The two largest viral ORFs (ORF 1a and ORF 1b) have been shown to encode the viral replicase (den Boon et al., 1991) and are located at the 5′-end of the viral genome between nucleotide positions 1 and 9807. ORFs 2 to 7 are overlapping and are situated at the 3′-end of EAV genome. The EAV transcript contains an N-glycosylated major membrane protein (“GL”, 30-44 kDa gene product of ORF 5), an unglycosylated membrane protein (“M”, 17 kDa gene product of ORF 6), and a phosphorylated nucleocapsid protein (“N”, 14 kDa, gene product of ORF 7). Moreover, the gene products derivable from the ORF 2 sequence include an N-glycosylated minor membrane protein (“GS”, from ORF-2b) and an envelope protein (“E”, from ORF-2a), whereby the latter protein was found to be conserved in all Arteriviruses (Snijder et al., 1999, Identification of a novel structural protein of arteriviruses. J. Virol. 73, 6335-6345).
An analysis of the genetic stability of EAV during horizontal and vertical transmission in an outbreak of EAV has revealed that the carrier stallion is the primary source of EAV genetic diversity (Balasuriya et al., 1999, Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis. J. Gen. Virol. 80, 1949-1958). It is known that the infected carrier stallion is a critical natural reservoir of EAV. EAV infection is maintained in horse populations primarily because chronic carrier animals shed the EAV in their semen, thus transmitting the virus during the mating process; the outbreak of an EAV infection can be initiated by the horizontal aerosol transmission of specific viral variants present in the semen fluid. One study has shown that not only does the carrier stallion act as the critical natural reservoir of EAV, but also that the genetic diversity of EAV is generated during the course of persistent infection in the infected horses (Patton et al., 1999, Phylogenetic characterization of a highly attenuated strain of equine arteritis virus from the semen of a persistently infected standardbred stallion. Arch. Virol, 144, 817-827).
The EAV ORF 6 encoded M protein is often considered to be the predominant target of the humoral immune response of horses against EAV. In one study, an enzyme linked immunosorbent assay (ELISA) was chosen to assess this response, and thus no biological function could be specifically attributed to the ensuing antibody response following exposure to EAV (Niewiesk et al., 1993, Susceptibility to measles virus-induced encephalitis in mice correlates with impaired antigen presentation to cytotoxic T lymphocytes, J. Virol. 67(1): 75-81). This M protein, together with the EAV ORF 5 encoded large envelope glycoprotein (GL), form the major EAV viral envelope protein, and are associated with each other via a heterodimer disulfide bridge.
Vaccination is a highly effective intervention useful for controlling infectious diseases. Immunization with a naked DNA sequence offers an attractive, relatively inexpensive and powerful vaccination approach. By using a eukaryotic expression vector harboring the DNA nucleic sequence of a specific antigenic determinant, live-attenuated, killed or peptide vaccines, it is possible to circumvent many of the undesirable side-effects associated with conventional vaccines. The endogenous production of antigen by the host cell transcription machinery mimics aspects of live attenuated vaccines without the associated potential risk of recombination with or reversion to wild-type virus. Furthermore, distinction between vaccine and wild-type pathogen may be difficult and time-consuming, since there are typically no marker gene(s) incorporated into live-attenuated vaccines, which is of particular concern in the field of veterinary medicine.
Immunization with plasmid DNA expressing foreign antigens may provoke both a cellular and humoral immune response, which provides optimal protection against most virally caused infectious diseases. Cytotoxic T-lymphocyte (“CTL”) activity is responsible for the elimination of infected cells, while antibodies bind to free virus to mediate lysis of infected cells. Viruses can, however, surreptitiously evade attack by the immune system and may establish persistent infections. Such viral persistence can result from several mechanisms, including high genetic variability of viral genomes, interference with cellular functions or depletion of subsets of immunocompetent cells.
Until now, attempts to develop a recombinant EAV vaccine therapy have been focused on the use of the EAV-derived M (ORF 6) and GPL (ORF 5) proteins, since: these proteins represent important targets for inducing a potent humoral immune response, are predominantly expressed on the viral outer membrane, and quantities of up to 30% of the EAV viral genome are usual.
One set of experiments has shown that an EAV vaccine composition consisting of the combination of EAV ORFs 2, 5, and 7 together, could successfully induce a stable and long-lasting immune response (Giese et al., 2002, Stable and Long-Lasting Immune Response in Horses after DNA Vaccination against Equine Arteritis Virus, Virus Genes, 25(2):159-67). The use of all three of these antigens in a combination vaccine (ORF 2: minor glycoprotein GPs; ORF 5: large envelope glycoprotein GPL; and ORF 7: nucleocapsid protein N) significantly stimulated cellular immunity in an antigen specific manner. While the EAV ORF 5 encoded viral membrane protein has been shown to be a powerful immunogen promoting humoral immunity; and the ORF 7 encoded viral capsid protein provokes a powerful cytotoxic response; the ORF 2 protein was included in the above EAV vaccine combination merely because it is highly conserved in the Arterivirus family and was thought to act as a stable back-up immunogen to the relatively unstable ORF 5, which has a very high mutation rate, despite the fact that the EAV ORF genome contains an extremely low percentage of the ORF 2 nucleic acid sequence (e.g. 1-2%), and therefore was not expected to itself generate any kind of meaningful immune response within the EAV vaccine.
Significantly, the use of EAV ORF 2 individually, i.e. without the other EAV ORF sequences, has not been the subject of any known EAV diagnostic or vaccine therapies, because relatively little is understood about the immunogenicity of this viral envelope glycoprotein. Moreover, as noted above, the EAV ORF 2 encoded proteins comprise only about 1-2% of the entire EAV virus. This particularly low antigen concentration could explain the rather poor antigenic recognition of the ORF 2 antigen in at least one mouse B-cell model, where limited antibody response was shown (Chirnside et al., 1995, Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL, J. Gen. Virol. 76, 1989-1998). These observations may explain why, until now, the exclusive use of ORF 2 in a vaccine composition against EAV has not been pursued.
The prevalence and highly contagious nature of EAV underscores the importance of finding preventative and/or therapeutic measures against this virus in order to prevent disastrous economic consequences on horse farming worldwide. Moreover, the development of an efficient vaccine composition, and methods of use, is of particular importance since these efforts contemplate both the preventative and therapeutic aspects of this disease.
Therefore, there is a need for an effective and ‘uncomplicated’ (e.g. low concentrations of antigen, low cross-reactivity, highly potent) vaccine composition against EAV that is capable of preventing and/or treating an EAV-associated disease, and a method of applying such a composition to a preferably equine subject.